The rapid diagnosis of viral infections is becoming an integral part of good medical practice. Some viruses have definable antigens against which antibodies can be produced. Therefore, immunoassays have been widely used for the measurement of the antigen and thus the determination of the presence or absence of a virion. There are cases, however, where antibody-antigen reactions are too specific, and the test may not reliably diagnose a viral infection. Where it is desirable to measure a more broad group of virions, it may be possible to detect a particular structural component of the virus. For example, influenza viruses possess surface glycoproteins that have neuraminidase activity. The surface glycoproteins hydrolyze substrates that contain .alpha.-ketosidically linked N-acetylneuraminic acid. When a virion with neuraminidase activity is incubated with a chromogenically or fluorometrically modified N-acetylneuraminic acid substrate, the enzyme will cleave the chromogenic or fluorometric group from the substrate and the reaction product will indicate the presence of the virion. The enzyme, in the case of influenza is part of the virus itself, but in other cases may be produced by the virus or by cells as a direct result of the cells being infected with the particular virus.
An assay for the direct measurement of influenza neuraminidase was developed by Yolken et al., J. Infectious Diseases 142:516-523 (1980). Yolken used the 4-methylumbelliferyl-.alpha.-ketoside of N-acetylneuraminic acid as a fluorescent substrate to measure neuraminidase activity in preparations containing small quantities of cultivated influenza virus as well as in some nasal wash specimens from human volunteers experimentally infected with influenza virus. Yolken suggested that "successful development of fluorometric enzyme assays for the detection of influenza neuraminidase might thus provide for a practical means of influenza diagnosis that is sufficiently rapid to allow for the institution of appropriate preventative and therapeutic interventions". Id. at 522. Yolken attempted detection with colorimetric neuraminidase assays. According to Yolken, the colorimetric assays were insufficiently sensitive for clinical applications. In fact, "visual neuraminidase substrates did not detect neuraminidase in any nasal wash specimen" that Yolken studied. Id. at 520. In contrast, Yolken noted that fluorometric assays may be suitable for detecting influenza neuraminidase in clinical samples. "The successful development of fluorometric enzyme assays for the detection of influenza neuraminidase might thus provide for a practical means of influenza diagnosis that is sufficiently rapid to allow for the institution of appropriate preventative and therapeutic interventions." Id. at 522. Following up on this hypothesis, Pachucki et al., J. Clinical Microbiology 26:2664-666 (1988), tested the 4-methylumbelliferyl-.alpha.-ketoside of N-acetylneuraminic acid on clinical specimens collected from influenza patients. They reported that, due to its low sensitivity, the "assay was not useful in detecting neuraminidase directly in clinical specimens but identified 91% of virus-positive isolates 24 h after inoculation onto tissue culture". Id. at 2665.
The combined teachings of these references, therefore, lead away from the use of neuraminidase substrates to detect influenza neuraminidase activity in clinical samples, and further teach that visual detection is impractical for any specimen.